Our long-term goal is to define how lysosome activity is regulated and to use this understanding to develop strategies to modulate lysosomal function for therapeutic purposes. Lysosomes perform critical functions with regard to the cellular degradation of macromolecules and the recycling of the nutrients that are liberated by this process. The transcription factor TFEB has been identified as a major regulator of genes encoding lysosomal proteins. Thus, the signaling mechanisms that regulate TFEB activity could contribute to the overall control of lysosome homeostasis. This project focuses on the role played by folliculin, the Birt-Hogg-Dub syndrome gene, and its binding partner folliculin interacting protein 1 (FNIP1) in this process. The proposed research builds on our novel observations that FLCN and FNIP1 localize to lysosomes and have a strong influence on regulating the nuclear levels of TFEB. Both the AMPK and mTORC1 signaling pathways that are related to cellular energy and nutrient homeostasis respectively may play a role in this action of FLCN and FNIP1. Our proposed research seeks to understand: 1) the role for TFEB as a FLCN and FNIP1 effector in the regulation of lysosome function; 2) the basis for the recruitment of folliculin and FNIP1 to lysosomes; 3) the physical interactions between FLCN, FNIP1 and AMPK and their effects on the recruitment of this kinase to the surface of lysosomes; and 4) the FLCN and FNIP1-dependent regulation of mTORC1 activation at the cytoplasmic surface of lysosomes. These studies will take advantage of our expertise in live cell imaging, protein-protein and protein-membrane interactions to analyze the dynamic recruitment of FLCN, FNIP1 and their binding partners to the cytoplasmic surface of lysosomes and the relationship of such recruitment to their function. This research builds on a growing appreciation of the role played by degradation and recycling of lysosomal substrates for meeting the energy and nutrient demands of cells and also highlights possible opportunities for the enhancement of lysosome function that could be relevant for the treatment of neurodegenerative diseases.